Staphylococcus aureus is an important emerging human pathogen responsible for both nosocomial infections and, increasingly, community acquired disease. S. aureus strains cause a variety of disease symptoms due to the production of a wide array of toxins. The emergence of strains resistant to multiple antibiotics, in combination with the horizontal transfer of toxin genes, poses a significant public health hazard. A large number of staphylococcal superantigens, including enterotoxins B, C and toxic shock toxin, have recently been shown to be encoded on a family of related, 15-20 kb pathogenicity islands (SaPIs). The SaPIs represent a novel type of mobile genetic element, which can be transferred at high frequency following helper phage infection, not only between S. aureus strains but to other bacterial pathogens as well. This high frequency transduction involves a remarkable example of molecular piracy, in which the SaPIs not only hijack the virion of the helper phage but direct the formation of smaller capsids which can accommodate the smaller SaPI genome while excluding that of the helper phage. In addition, the SaPIs redirect the specificity of the phage DNA packaging machinery so that SaPI DNA is preferentially encapsidated. Recent studies have identified some of the SaPI and helper phage genes that play a role in SaPI mobilization and SaPI interference with helper phage growth, but molecular details remain to be elucidated. This proposal includes a combination of genetic, biochemical and structural approaches to address aspects of the SaPI-helper interaction, with the following specific aims: (1). Characterize the roles of the genes involved in capsid formation and the mechanism for SaPI capsid size determination. (2). Characterize the determinants for SaPI redirection of DNA packaging. These studies will provide insight into the novel relationship between two accessory genetic elements that are responsible for the dissemination of staphylococcal enterotoxins and contribute to our understanding of the evolution of virulence in this important pathogen and the intergeneric transfer of virulence determinants in microbial communities. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus has become a major health problem in hospitals, especially with the emergence of multiple antibiotic resistant strains, and deaths attributable to Staphylococcus aureus infections have recently surpassed those from AIDS. Pathogenic S. aureus can cause severe systemic infections by producing several toxins from genes carried on so-called pathogenicity islands in the bacterial genome. Understanding the mechanism by which these pathogenicity islands are mobilized by staphylococcal bacteriophages will assist in the development of strategies to inhibit the transfer of toxin genes between S. aureus strains and help slow the emergence of highly virulent, multiresistant organisms.